MICROSCOPY Vol.43▶No.2 2008
â– Researches Today

Application of SDS-digested freeze-fracture replica labeling for molecular localization in neural tissues

Yugo Fukazawa and Ryuichi Shigemoto

Abstract: Identification of functionally differentiated structural specializations in the plasma membrane and precise localization of functional molecules in the specialization is crucial in understanding the intercellular signaling mechanisms. SDS-digested freeze-fracture replica labeling (SDS-FRL) was innovated as a suitable technique to reveal the fine structure of plasma membrane and quantitative localization of biomolecules over the membrane structure. However, it was difficult to apply this technique to the tissues having multiple types of cell populations due to two reasons. One reason is the poor morphological clues in the replicated membrane in order to identify the cellular origin of observed profiles. Another is the difficulty in reproducibility of the quantification results due to uneven removal of tissue by SDS treatment from the replica membrane. Here, we introduced our current SDS-FRL protocol, which is optimized for the precise localization and quantitative investigation of the plasma membrane molecules on neural tissue. We also elaborated several technical clues to overcome above mentioned problems. Hopefully, this information can provide help to those who are interested in employing this technique.

Key words: Freeze-fracture replica, High-pressure freezing, Plasma membrane, Ultrastructure, Immunolabeling